Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis

Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING domain of rat RNF4 in complex with E2 (UbcH5A) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The carboxy-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilize the consequent tetrahedral transition-state intermediate. This study presents the crystal structure of a RING-type E3 ligase bound to ubiquitin-loaded E2; the structure reveals how ubiquitin binding to E2 leads to changes in the catalytic site, priming it for catalysis by the E3 enzyme. RING E3 ligase primed for ubiquitin transfer Post-translational modification of proteins by ubiquitin and its homologues is important for many physiological processes. Ubiquitin is transferred to target proteins from a thioester complex with a ubiquitin-conjugating enzyme (E2) in a step catalysed by a ubiquitin ligase (E3). In this study, Ronald Hay and colleagues present the crystal structure of a RING type E3 ligase bound to ubiquitin-loaded E2. The structure reveals how ubiquitin binding to E2 leads to changes in the catalytic site, priming it for catalysis by the E3 enzyme. The mechanisms proposed here are likely to be conserved among other ubiquitin-conjugating enzymes.